SIGNIFICANCE: mFluiDx is developing tests for a target pathogen occurring frequently in primary care settings that is difficult to differentiate by observing symptoms alone. Culturing takes too much time, and serology has poor sensitivity. PCR is significantly more accurate; however, it is too costly, time-consuming, and complex for primary care clinics to adopt. Difficult diagnosis often compels multiple patient visits, increasing hospitalization risk due to delayed treatment. As automated NAAT solutions remain too costly, there are currently no point-of-care nucleic acid amplification tests (POC-NAAT) ideal for primary care testing of mFluIDx?s target pathogen. Successful development of a POC-NAAT can help prevent hospitalization, reduce misuse of antibiotics, and improve patient quality of life, all while saving time and medical resources. INNOVATION: mFluiDx is developing vacuum microfluidic technologies ? a new class of microfluidics that enables automated, highly multiplexed, quantitative NAAT at 100X lower cost than conventional qPCR setups. SA1 Design primer/probe sets for rapid isothermal amplification: mFluiDx will develop primers and probes for detecting the target pathogen based on a rapid isothermal amplification chemistry. SA2 Design a low-cost, automated microfluidic nucleic acid sample preparation module for vacuum microfluidics: Nucleic acid extraction is a major bottle-neck that hinders POC NAAT deployment. Robotic systems are very costly. Conventional spin-columns kits require extra manual labor. mFluiDx will develop a sample prep module, which enables one-step sample preparation. This will be the first sample preparation module designed for vacuum microfluidics, with a focus on low cost and automation. SA3 Design a vacuum-microfluidic readout chip: mFluiDx will design a vacuum microfluidic module downstream of the sample prep module. This vacuum microfluidic module serves as a location for multiplexed isothermal amplification and optical readout. SA4 Proof-of-concept one-step NAAT with 100X less setup cost than qPCR: mFluiDx will demonstrate one-step NAAT from spiked samples after integrating the sample prep module with the vacuum readout chip in SA2 and SA3. Successful development of mFluiDx?s platform will be the first proof-of-concept of a POC NAAT for mFluiDx?s target pathogen, specifically designed to be automated and low-cost enough for primary care settings. Down the road, this platform can perform multiplexed NAAT for other viral/bacterial infections as well. A Phase II proposal will focus on scalable manufacturing design, completing an optical reader, and clinical performance studies. Results will be used for a FDA 510(K) application.